Aminomalonyl alanine compounds as dietary sweeteners

ABSTRACT

Compounds having relatively long shelf lives in acidic aqueous media and which are useful as sweeteners have the formula ##STR1## or are pharmaceutically acceptable salts thereof, wherein X is --OR or --NHR, or --NH 2 , with R being alkyl having 3-10 carbon atoms.

FIELD OF THE INVENTION

This invention relates to new chemical compounds and to methods of usingthem as dietary sweeteners. In particular the invention also relates tocompounds broadly classified as aminomalonyl D-alanine derivatives.

BACKGROUND OF THE INVENTION

Since the introduction of saccharin as an artificial sweetener, relativefew new sweeteners have been developed. Among those that have beendiscovered, however, chief among them is the methyl ester ofL-α-Aspartyl-L-phenylalanine, more commonly known as aspartame,disclosed in U.S. Pat. No. 3,492,131 to Schlatter. The viability ofaspartame's use in non-dry applications is in serious question, however.Its recent introduction for use in soft drinks in this country was as amixture with saccharin, the saccharin being used to maintain a sweettaste long after aspartame hydrolyzes to a non-sweet structure.

Thus, a non-toxic artificial sweetener compound comparable in sweeteningability to aspartame but which exhibits superior stability to aspartamein aqueous media would be a useful addition to the artificial sweetenersindustry. Such compounds, compositions containing them, and methods ofusing these compounds as artificial sweeteners are the subject of thepresent invention.

Japanese Pat. No. 28068 (Takeda) describes aminomalonyl dipeptides whichhave a C-terminal amino acid of the L-configuration as sweeteningcompounds for preparing food. Surprisingly, we have found that such isnot the case, but rather that the corresponding compounds of the presentinvention having a C-terminal amino acid of the D-configuration aresweet.

SUMMARY OF THE INVENTION

The present invention provides compounds useful as artificial sweetenershaving the following structure: ##STR2## wherein X or --OR or --NHR, Rbeing alkyl of 3-10 carbon atoms, or --NH₂. A preferred embodimentresults when X or --OR and R is an isopropyl group, i.e. the isopropylester of aminomalonyl-D-alanine (herein also referred to simply as the"isopropyl ester"). Stereochemically, when synthesized the compounds ofthe present invention result as diastereomeric mixtures. The aboveformula is shown for convenience but it should be noted that thecompounds will likely exist as zwitterions.

The pharmaceutically acceptable salts, such as the citrate, tartrate,hydrochloride, and phosphate, of the compounds disclosed herein are alsoeffective as sweeteners. Such salts can be made using typical acids andprocedures for making the salts are as conventionally known andpracticed in the art.

The compounds of this invention generally range in sweetness betweenthat of sucrose and aspartame. For example, the isopropyl ester ofaminomalonyl alanine is 58 times sweeter than sucrose and about half assweet as aspartame on a weight basis. Compared to saccharin, theisopropyl ester does not possess a "metallic" aftertaste, and there arevirtually no detracting side tastes.

Additionally, the compound of this invention are believed to benon-toxic. Concerns have been raised regarding the principal metabolismproducts of aspartame--aspartic acid and phenylalanine. The formercauses brain lesions in neonatal mice and the latter has been reportedto induce grand mal-type seizures in monkeys, produce birth defects inpregnant women with phenylketonuria, induce behavioral changes, andalter brain chemistry. Saccharin itself is well known as a weak mutagenin the Ames assay, a property which is associated with carcinogenicpotential.

In addition to the compounds having the structure given above, theinvention also provides a method of sweetening a beverage, comprisingdissolving a sufficient amount of at least one of the compoundsdisclosed herein to effect said sweetening.

It is therefore an object of this invention to provide an artificialsweetener.

It is further an object of this invention to provide an artificialsweetener which is relatively stable in aqueous solution.

It is further an object of this invention to provide an artificialsweetener which is non-toxic.

These and other objects and advantages of the invention will become moreapparent and more readily appreciated from the following detaileddescription of presently preferred exemplary embodiments of theinvention taken in conjunction with the accompanying FIGURE which is adiagrammatic representation of the synthesis ofDL-aminomalonyl-D-alanine isopropyl ester. The lower case letterdesignations refer to the following:

a. Benzyl chloroformate (Z--Cl), NaHCO₃

b. NaOH, aq. EtOH

c. DBU, Benzyl bromide

d. tetramethylguanidine (TMG), aq. tetrahydrofuran (THF)

e. DCC, HOBt, dimethylformamide (DMF), D-alanine isopropyl ester (6)

f. 10% Pd/C, H₂, MeOH.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart illustrating the synthesis ofDL-aminomalonyl-D-alanine isopropyl ester.

FIG. 2 is a graphical comparison of the sweetness potency of theisopropyl ester with sucrose and aspartame. Each data point representsthe mean∓SEM.

FIG. 3 is a graphical comparison of the sweetness potency of theD-isopropyl ester with the corresponding L-isopropyl ester and sucrose.

DETAILED DESCRIPTION

The synthesis of compounds within the scope of this invention will bedescribed. This description is strictly for purposes of illustration,not limitation.

I. Synthesis of DL-aminomalonyl-D-alanine isopropyl ester.

As an overview of the synthetic methods employed, the synthesis ofDL-aminomalonyl-D-alanine isopropyl ester is shown in FIG. 1 and is nowbriefly summarized. Commercially available aminomalonic acid diethylester hydrochloride 1 was treated with Z-Cl (Z is an abbreviation for abenzyloxycarbonyl group) and NaHCO₃ to afford the N-protectedZ-Ama-(OEt)₂ 2 (Ama=aminomalonyl group; Et=ethyl). Saponificationprovided the free acid 3. Treatment with excess benzyl bromide and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) yielded the diester 4 whichupon partial saponification provided the monobenzyl ester 5.

Coupling of 5 with D-alanine isopropyl ester 6 to afford the protecteddipeptide 7 was achieved by standard dicyclohexylcarbodiimide (DCC)mediated procedures employing either hydroxybenzotriazole (HOBt) orN-hydroxysuccinimide (HOSu) as catalysts. The yields with eithercatalyst were comparable (70-80%), with the latter allowing easierremoval of the reaction solvent (THF vs DMF) required for solubilityreasons.

The final step in the sequence, removal of the benzyloxycarbonyl (Z) andbenzyl (Bzl) moieties by hydrogenolysis over 10% Pd on carbon inmethanol was followed by recrystallization from methanol to affordDL-aminomalonyl-D-alanine isopropyl ester.

Intermediates 2, 4, 5, and 6 are known compounds. The conversion of 4 to5 using tetramethylguanidine is also believed to be novel.

Detailed procedural steps may be described as follows.

N-Benzyloxycarbonyl-aminomalonic Acid Diethyl Ester (2)

This compound was prepared according to the procedure of M. Fujino etal, Chem. Pharm. Bull. Japan, 24 (9), 2112 (1976), in which it wasreported as an oil. The present inventors crystallized the crude oilfrom ether-petroleum ether in a dry ice-acetone bath, with scratching,to yield 5.13 g (83%); mp 37°-38° C.; 60 MHz nmr(CDCl₃, ppm) 1.25 (6H,t, J=7 Hz, CH₃ CH₂ --), 4.20 (4H, q, J=7 Hz, CH₃ CH₂ --), 5.03 (2H, s,ArCH₂ --), 5.66 (1H, broad s, amide H), 7.23 (5H, s, ArH₅).

N-Benzyloxycarbonyl-aminomalonic Acid (3)

Compound 2 (3.49 mmol, 1.08 g) was dissolved in 3 mL 95% ethanol andtreated with 3.49 mL 2N NaOH. The clear, colorless solution was left atroom temperature for 19 hours until no starting material could bedetected by tlc on silica gel (benzene:ethyl acetate, 4:1). Ethanol wasevaporated under reduced pressure at room temperature. The aqueoussolution was washed once with ether and then acidified with concentratedHCl to pH 2.5. The milky solution was extracted with ethyl acetate (3x).The pooled extract was washed with brine, dried over anhydrous Na₂ SO₄and evaporated to dryness. The product was crystallized from ether toyield 0.852 g of compound 3; mp 144°-145° C.; homogeneous by the tlc onsilic gel (CHCl₃ :methanol:acetate acid, 5:13:1); 250 MHz nmr(acetone-d₆, ppm) 5.00 (1H, d, J=8 Hz, α--H), 5.10 (2H, s, ArCH₂ --),6.84 (1H, d, J=8 Hz, amide H), 7.36 (5H, m, ArH₅). In a 60 MHz spectrumrun in low water content acetone, a 9.7 ppm (2H, broad s, exchangeablewith D₂ O) was also observed.

N-Benzyloxycarbonyl-DL-aminomalonic Acid Monobenzyl Ester (5)

Compound 3 (4.55 mmol, 1.25 g) was dissolved in a mixture of 8 mLtetrahydrofuran (THF) and 8 mL CH₃ CN. Benzyl bromide (9.88 mmol, 1.18mL) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (N. Ono et al., Bull.Chem. Soc, Japan, 51, 2401, 1978; C. G. Rao, Org. Prep. Proc. Int., 12,225, 1980) (9.88 mmol, 1.48 mL) were added. The clear, colorlesssolution was left at room temperature overnight. The solution wasconcentrated under reduced pressure to half of the original volume. Theproduct was precipitated by addition of water. The white crystallinematerial was collected by filtration, dried and triturated withpetroleum ether to yield compound 4; 1.72 g (80%); mp 109°-110° C.; 250MHz nmr (CDCl₃, ppm), 5.11, 5.16, 5.2 (7H, s, m, 3 ArCH₂ -- and α--H),5.82 (1H, d, J=8 Hz, NH), 7.30 (15H, m, 3 ArH₅).

Z-Ama(OBzl)₂ (1.78 g, 4 mmol) was dissolved in 18 mL THF. TMG (0.5 mL, 4mmol, freshly distilled) in H₂ O was added. The clear colorless solutiongive a pH of 12.5-13.5 (pH hydrion paper). After 3 h at room temperaturethe pH went down to 8.5. The solution was evaporated under reducedpressure to remove THF. The cloudy solution was diluted with more waterand extracted with ether. The aqueous phase was acidified to pH 2 withconcentrated HCl. The cloudy aqueous phase was extracted with EtOAc(3×). The pooled EtOAc extract was washed with H₂ O, dried over Na₂ SO₄and evaporated to dryness to give a white solid. The crude product wasrecrystallized from MeOH--H₂ O. Yield: 1.17 g (86%), m.p. 118°-120° C.,tlc on silica gel [CHCl₃ :MeOH:HOAc=45:5:1] indicated the absence of 3and 4. 250 MHz nmr (acetone-d₆, ppm). 5.06 (1H, d, partially overlappedwith 5.1 peak, α--H), 5.1 (2H, s, ArCH₂ --), 5.24 (2H, s, ArCH₂ '--),7.0 (1H, broad s, amide H), 7.35 (10H, m, 2 ArH₅).

N-Benzyloxycarbonyl-DL-aminomalonyl(benzyl ester)-D-alanine IsopropylEster (7)

N-Benzyloxycarbonyl-DL-aminomalonic acid monobenzyl ester 5 (0.156 g,0.455 mmol) was dissolved in 1.5 mL DMF. The solution was cooled to -15°to -20° and then treated with DCC (94 mg, 0.455 mmol) and1-hydroxybenzotriazole hydrate (62 mg, 0.46 mmol). The reaction mixturewas stirred at -15° to -20° for 30 minutes. D-Alanine isopropyl esterhydrochloride (69 mg, 0.46 mmol) was then introduced, followed by theaddition of N-methylmorpholine (50 microliters, 0.46 mmol). The reactionmixture was stirred at -15° to -20° C. for 1.5 hr, at 0° for 1 hr and 8hr at room temperature. The reaction mixture was filtered to remove DCUand the filtrate was evaporated to dryness under high vacuum. The oilyresidue was taken up in EtOAc and the cloudy EtOAc solution wasfiltered. The EtOAc filtrate was then washed with 0.1N HCl (3×), H₂ O(3×), 0.1N NaOH (3×), H₂ O (3×), dried over anhydrous Na₂ SO₄ andevaporated to dryness to give a clear oil. TLC on silica gel(benzene:EtOAc=4:1 and toluene:EtOAc=9:1) each indicated 3 spots; yield0.187 g (91%). The crude material (0.167 g) was dissolved in CHCl₃,mixed with approximately 0.2 g of silica gel and loaded on a 12 g silicagel column in toluene. The crude product was then chromatographed bygradient elution with toluene to toluene:EtOAc=8:1. The major fraction(0.124 g) was identified by nmr as the desired product. The product wasdissolved in a minimum amount of warm EtOAc; the solution was thendiluted with an equal volume of ether and refrigerated. A white solidwas obtained: mp 125°-130°, 250 MHz nmr (CDCl₃, ppm), 1.23 (m, 7.5H,(CH₃)₂ --CH--O-- overlapping with β--CH₃ of Ala of one of the 2diastereomers), 1.37 (d, 1.5H, J=6.98 Hz, β--CH₃ of Ala of the seconddiastereomer, 4.45 (m, 1H, α--H of Ala), 5.00 (m, 1H, α--H ofisopropyl), 5.12 (s, 2H, Ar--CH₂ --), 5.18-5.34 (m, 3H, Ar--CH₂ --O-- ofthe benzyl ester overlapping with α--H of Ama), 7.33 (s, 10H, 2 ArH₅).

A 30 mmol scale preparation following the above procedure through theextraction step afforded the crude product which was crystallized from2-propanol to give 8.91 g of tlc homogeneous 7. Another 2.17 g (totalyield 81%) of 7 was obtained from the mother liquor by the above columnchromatographic procedure.

Anal. Calcd for C₂₄ H₂₈ N₂ O₇ : C, 63.46; H, 6.18; N, 6.14; Found: C,63.15; H, 6.04; N, 6.06.

DL-Aminomalonyl-D-Alanine Isopropyl Ester (8)

N-Benzyloxycarbonyl-DL-aminomalonyl(monobenzyl ester)-D-alanineisopropyl ester 7 (1.16 g, 2.53 mmol) was hydrogenated at roomtemperature in 100 mL MeOH over 0.573 g of 10% Pd/C at 40 psi in a Parrapparatus for 1 hr. The solution was then filtered through a Celite padto remove the catalyst; the filtrate was concentrated under reducedpressure and the free dipeptides were precipitated from the solution byaddition of ether. The crude product was recrystallized from MeOH. Yield0.411 g (60%), mp 102°-104°, 250 MHz nmr (CD₃ OD, ppm) 1.25 (m, 6H,(CH₃)₂ CH--O--), 1.41 (2d, 3H, β--CH₃ of Ala), 4.38 (2q, 1H, α--H ofAlA), 4.99 (m, 1H, α--H of isopropyl).

Anal. Calcd for C₉ H₁₆ N₂ O₅ : C, 46.55; H, 6.95; N, 12.06; Found C,46.53; H, 6.95; N, 12.11.

L-Alanine Iopropyl Ester Hydrochloride (9)

L-Alanine (10 g, 0.112 mole) was suspended in 250 mL 2-propanol.Hydrogen chloride gas was bubbled into the solution. The temperature ofthe solution gradually rose to above 70° C. as hydrogen chloride wasdissolved in the alcohol. After 21/2 h, most of the free amino acid wentinto solution; more 2-propanol was added in small portions(approximately 30-50 mL) until a clear solution was obtained. Thesolution was left at room temperature overnight. The solution wasevaporated through a NaOH trap under reduced pressure. The residue wasdissolved in 100 mL 2-propanol and again evaporated to dryness. Theresidue was dried under high vaccum and a white mass was obtained, whichwas triturated with ether and collected by filtration to yield 16.4 g(96%), mp 84°-87° C. The crude product was recrystallized from CH₃CN--ether: mp 89°-90° C.; nmr (CD₃ OD, ppm), 1.28 (d, J=6 Hz, 6H, CH₃ ofisopropyl), 1.51 (3H, d J=8 Hz, β--CH₃), 3.98 (1H, q, J=7 Hz, Ala α--H),4.7 (3H, broad s, amino H), 5.0 (1H, m, O--CHMe₂). The D-isomer wassimilarly prepared.

N-Benzyloxycarbonyl-D-alanine trans-2-Methylcyclohexyl Ester (10)

N-Benzyloxycarbonyl-D-alanine (2.5 mmol. 0.556 g) andtrans-2-methylcyclohexanol (3 equivalents, 1 mL) were dissolved in 7 mLTHF at room temperature, followed by the addition of 70 mg4-dimethylaminopyridine. DCC (2.5 mmol, 0.515 g) was then introducedwith stirring. The solution turned cloudy immediately and was stirred atroom temperature overnight. The reaction mixture was stirred for another15 min after several drops of HOAc were added to it. Dicyclohexylurea(DCU) was filtered off and the filtrate evaporated to dryness. Theresidue was dissolved in ethyl acetate. The EtOAc solution was filtered,washed with 0.5N HCl (2×), brine (2×), 5% NaHCO₃ (2×) and finally brine(2×). The organic phase, after being dried over anhydrous Na₂ SO₄,showed one UV-positive tlc spot. A second spot, which could only bedetected by phosphomolybdic acid (PMA)--Ce(SO₄)₂ spray, was identifiedas the unreacted alcohol by tlc comparison with authentic material. Theethyl acetate solution was evaporated to dryness and dried under highvacuum, giving a clear oil weighing 0.965 g. The crude material waschromatographed on a Merck Si--60 size C prepacked column by gradientelution with toluene to toluene:ethyl acetate (9:1). After evaporation,0.629 g (72.0%) of clear, colorless oil was recovered: nmr (CDCl₃, ppm),0.85-2.05 (13H, m, aliphatic H of cyclohexanol), 0.9 (3H, d, J=6 Hz,CH₃) 1.3 (3H, d, J=7 Hz, obscured by other aliphatic protons, β--CH₃ ofAla), 4.3 (2H, m, CH--O-- and α--H), 5 (2H, s, ArCH₂ --), 5.25 (1H,broad s, amide H), 7.15 (5H, s, ArH₅).

N-Benzoyloxycarbonyl-D-alanine Fenchyl Ester (11)

N-Benzyloxycarbonyl-D-alanine (8.646 g, 38.7 mmol) was dissolved in 150mL THF and treated with DCC (7.987 g, 38.7 mmol) and 15 g of fenchylalcohol (2.5 eq), followed by 4-dimethylaminopyridine (1.1 g). Thereaction mixture was stirred at r.t. overnight. Several drops of HOAcwere added and after 30 min DCU was filtered off. The filtrate wasevaporated to dryness under reduced pressure and the oily residue wasdissolved in EtOAc. The solution was refrigerated and more DCU wasfiltered off. The EtOAc solution was washed with 0.1N HCl (3×), H₂ O,0.1N NaOH (3×), H₂ O (3×) and dried over Na₂ SO₄. The solution was thenevaporated to dryness under reduced pressure. The crude oily product soobtained was subjected to sublimation at 60° C./0.8 mm for 5 hours untilthe weight of the crude product was approximately 12-13 g. The oil wascrystallized from hexane: yield 9.75 g (70%). Tlc (reverse phase C-18plate, MeOH: H₂ O=8:1) showed a single PMA--Ce(SO₄)₂ positive spot, m.p.97°-98°. An analytical sample was recrystallized from ether, m.p.98°-99°, 250 MHz nmr (CDCl₃, ppm) 0.76 (s, 3H, fenchyl 1-methyl), 1.04(s, 3H, fenchyl 3-methyl), 1.08 (s, 3H, fenchyl 3-methyl), 1.12-1.79 (m,7H, fenchyl aliphatic protons), 1.40 (d, 3H, J=7.35 Hz, β--CH₃ of Ala),4.24 (q, 1H, J=7.35 Hz, α--H of Ala), 4.35 (d, 1H, J=1.8 Hz, α--H offenchyl alcohol). Anal. Calcd for C₂₁ H₁₉ NO₄ : C, 70.16; H, 8.13; N,3.89; Found C, 70.05; H, 7.98; N, 3.78.

D-Alanine Fenchyl Ester Hydrochloride (12)

Crystalline N-benzyloxycarbonyl-D-alanine fenchyl ester (9.1 g, 25.3mmol) was hydrogenated in 120 mL absolute MeOH and 22 mL 1.25N HCl/etherover 1.64 g of 10% Pd/C at 38 psi for 30 min in a Parr apparatus. Thecatalyst was removed by filtration through a Celite pad and the filtrateevaporated to dryness under reduced pressure. The salt was crystallizedfrom ether. Yield: 5.68 g (86%) in 2 crops. mp 180°-181°, 250 MHz nmr(acetone-d₆, ppm) 0.85 (s, 3H fenchyl 1-methyl), 1.08 (s, 3H, fenchyl3-methyl), 1.11 (s, 3H, fenchyl 3-methyl), 1.14-1.89 (m, 7H, fenchylaliphatic protons), 1.74 (d, 3H, J=7.35 Hz, β--CH₃ of Ala), 4.35 (q,J=7.35 Hz, α--H of Ala), 4.43 (25, 1H, α--H of fenchyl alcohol).

D-Alanine trans- 2-Methylcyclohexyl Ester p-Toluenesulfonate (13)

N-Benzyloxycarbonyl-D-alanine-trans-2-methylcyclohexyl ester (14.7 g,47.5 mmol) and p-toluenesulfonic acid monohydrate (9.04 g, 47.5 mmol)were dissolved in 135 mL MeOH. The methanolic solution was hydrogenatedover 2.51 g of 10% Pd/C at 40 psi in a Parr apparatus for 1 hour. Thecatalyst was removed by filtration through a Celite pad and the filtratewas concentrated under reduced pressure. The salt was precipitated byaddition of ether. Yield: 13.8 g in 2 crops (81%) mp 145°-147°. Tlc onsilica gel (CHCl₃ :MeOH:HOAc=45:5:1) indicated a single ninhydrinpositive spot. 250 MHz nmr (CD₃ OD, ppm) 0.92 (d, 3H, J=6.25 Hz,trans-2-methyl), 1.09-1.99 (m, 7H, aliphatic cyclohexyl protons), 1.53(2d, 3H, β--CH₃ of Ala), 2.37 (s, 3H, CH₃ --Ar--SO₃ H), 4.09 (2q, 1H,J=7.35 Hz, 2.57 Hz; α--H of Ala), 4.51 (m, 1H, α--H of alcohol); 7.22,7.69 (ABq, 4H, J=8.46 Hz, CH₃ --ArH₄ --SO₃ H).

N-Benzyloxycarbonyl-D-alanine 1-Adamantyl Ester (14)

N-Benzyloxycarbonyl-D-alanine (11.15 g, 50 mmol) was dissolved in 250 mLTHF and treated with DCC (10.3 g, 50 mmol), 1-adamantanol (7.7 g, 51mmol) and 1.45 g of 4-dimethylaminopyridine. The reaction mixture wasstirred at room temperature. At the end of 24 hours, a few drops of HOAcwas added and the reaction mixture was stirred for an additional 15minutes. DCU was filtered off, the filtrate was evaporated to drynessunder reduced pressure. The oily residue so obtained was dissolved inEtOAc and the solution was refrigerated overnight. More DCU was filteredoff. The filtrate was then washed with 0.1N HCl (3×), H₂ O (3×), 0.1NNaOH (3×), H₂ O (3×) and dried over anhydrous Na₂ SO₄. The solution wasevaporated to dryness to give a clear oil. The oil was subjected tosublimation at 100°/0.1 mm for 5 hours. The oily residue wascrystallized from hexane: mp 72°-74°, yield 11.6 g (75%), 250 MHz nmr(CDCl₃, ppm) 1.37 (d, 3H, J=7.35 Hz, β--CH₃ of Ala), 1.65 (s, 6H,adamantyl aliphatic protons of C's 4, 6 and 10), 2.09 (s, 6H,1-adamantyl aliphatic protons of C's 2, 8 and 9), 2.16 (s, 3H,1-adamantyl aliphatic protons of C's 3, 5 and 7), 4.23 (m, 1H, α--H ofAla), 5.09 (s, 2H, Ar--CH₂ --O--), 5.32 (br.d, amide proton), 7.33 (s,5H, ArH₅).

D-Alanine 1-Adamantyl Ester Hydrochloride (15)

N-Benzyloxycarbonyl-D-alanine 1-adamantyl ester (7.35 g, 20.6 mmol) wasdissolved in 100 mL MeOH and 14.5 mL 1.25N HCl/ether. The solution washydrogenated over 1.09 g of 10% Pd/C at 40 psi in a Parr apparatus atroom temperature for 40 minutes. The solution was filtered through aCelite pad and the clear filtrate was evaporated to dryness to give ayellow oil, which was crystallized from EtOAc. A light yellow salt (4.2g) was obtained. The crude product was recrystallized from MeOH-EtOAc;yielded 7.85 g (72%), mp 218°-219° (dec), 250 MHz nmr (CD₃ OD, ppm) 1.50(d, 3H, J=7.35 Hz, β--CH₃ of Ala), 1.72 (m, 6H, 1-adamantyl aliphaticprotons of C's 2, 8 and 9), 2.18 (m, 9H, adamantyl aliphatic protons),3.95 (q, 1H, J=7.35 Hz, α--H of Ala).

N-Benzyloxycarbonyl-DL aminomalonyl(benzyl ester)-D-alanine FenchylEster (16)

N-Benzyloxycarbonyl-DL-aminomalonic acid monobenzyl ester (6.86 g, 20mmol) was dissolved in 40 mL DMF and cooled to -20° C. DCC (4.12 g, 20mmol) and 1-hydroxybenzotriazole hydrate (HOBt.H₂ O) (2.70 g, 20 mmol)were then added. The reaction mixture was stirred at -20° C. for 30minutes. D-Alanine fenchyl ester hydrochloride (5.23 g, 20 mmol) wasadded, followed by the addition of N-methylmorpholine (2.2 mL, 20 mmol).It was stirred at -20° to 15° C. for 1.5 hour, 0° C. for 2 hours andr.t. overnight. Several drops of HOAc were added and the reactionmixture was stirred for an additional 30 minutes. After DCU was removedby filtration, the filtrate was evaporated under high vacuum to dryness.The oil so obtained was dissolved in EtOAc and the solution wasrefrigerated. More DCU was filtered off. The EtOAc solution wasextracted with H₂ O (2×), 0.1N HCl (3×), H₂ O (3×), 0.1N NaOH (3×) andH₂ O (3×). The organic phase was dried over Na₂ SO₄ and evaporated todryness. The oil was crystallized from hexane. Yield 2.81 g, mp 71°-75°C., tlc on silica gel (benzene:EtOAc=9:1), single spot (Cl₂ +o-tolidinespray). Another 5.16 g of the product was isolated by chromatographingthe mother liquor (crude weight=8.32 g) on a column 360 g of silica gelusing toluene: EtOAc=7.1 as the eluant (overall yield=72.5%). 250 MHznmr (CDCl₃, ppm), 0.74 and 0.76 (2 s, 3H, diastereomeric, fenchyl1-methyl), 1.02 and 1.04 (2 s, 3H, diastereomeric fenchyl 3-methyl),1.10 and 1.11 (2 s, 3H, diastereomeric fenchyl 3-methyl), 1.18-1.73 (m,7H, fenchyl aliphatic protons), 1.43 (d, 3H, J=7.0 Hz, β--CH₃ of Ala,partially overlapped with fenchyl aliphatic protons), 4.55 (q, 1H, J=7.0Hz, α--H of Ala, 4.97 (2br s, 1H, α--H of fenchyl), 5.12 (s, 2H, Ar--CH₂--), 5.18-5.37 (m, 3H, Ar--CH₂ overlapped with α--H of Ama), 6.03 (br d,1H, amide proton), 6.83 and 7.04 (2 br d, 1H, urethane H), 7.33 (s, 10H,2 ArH₅).

N-Benzyloxycarbonyl-DL-aminomalonyl(benzyl ester)-L-alanine IsopropylEster (17) (intermediate for comparative non-sweet isomers)

This compound was prepared by the same DCCHOBt procedure as describedfor the D-alanine isomers on a 30 mmol scale. The crude product wascrystallized from 2-propanol and 7.25 g of tlc homogeneous material (onsilica gel toluene:EtOAc=4:1) was collected. The filtrate, showing 4spots on tlc in the same solvent system was chromatographed on silicagel by gradient elution with toluene to toluene:EtOAc=5:1. The majorfraction collected was crystallized from 2-propanol; yielded 2.76 g in 3crops; overall yield 10.0 g (73%), mp 130°-133°. 250 MHz nmr (CDCl₃,ppm) 1.23 (m, 7.5 H, (CH₃)₂ CH--O-- overlapping with β--CH₃ of Ala ofone of the 2 diastereomers), 1.37 (d, 1.5H, β--CH₃ of Ala of the seconddiastereomer), 4.45 (m, 1H, α--H of Ala), 5.0 (m, 1H, α--H ofisopropyl), 5.12 (s, 2H, Ar--CH₂ of Z). 5.18-5.34 (m, 3H, Ar--CH₂ --Ooverlapping with α--H of Ala), 6.02 (br d, H, amide H) 6.85 (br d, 1H,urethane H), 7.33 (s, 10H, 2 ArH₅). Anal. Calcd for C₂₄ H₂₈ N₂ O₇ : C,63.15; H, 6.18; N, 6.14; Found C, 62.98; H, 6.21; N, 5.96.

N-Benzyloxycarbonyl-DL-aminomalonyl(benzyl ester)-D-alaninetrans-2-Methylcyclohexyl Ester (18)

N-Benzyloxycarbonyl-DL-aminomalonic acid monobenzyl ester (0.343 g, 1mmol) was dissolved in 4 mL THF and cooled to -15° to -20°. DCC (0.206g, 1 mmol) and N-hydroxysuccinimide (0.115 g, 1 mmol) were then added.The reaction mixture was stirred in the cold for 30 minutes. D-Alaninetrans-2-methylcyclohexyl ester p-toluenesulfonate (0.358 g, 1 mmol) wasthen suspended in the solution, followed by the addition of Et₃ N (0.14mL, 1 mmol). The reaction mixture was stirred at -15° to -20° for 2.5hours, 0° for 1 hour and room temperature overnight. A drop of HOAc wasadded. The reaction mixture was stirred for an additional 15 minutes.After DCU was removed by filtration, the filtrate was evaporated todryness to give a clear oil. A white crystalline material was obtainedwhen the oil was triturated with water; yield 0.55 g; tlc on silica gel(toluene:EtOAc=4:1) gave 4 spots. The crude product was purified on 25 gsilica gel by gradient elution with toluene to toluene:EtOAc=5:1. Themajor fraction collected was identified by nmr as the desired dipeptide.The oil was crystallized by trituration in hexane: yield 0.391 g, mp116°-119°, 250 MHz nmr (CDCl₃, ppm) 0.87 (2 d, 3H, 2-methyl), 1.04-1.11(m, 9H, cyclohexyl aliphatic protons), 1.22 and 1.40 (m, 3H, β--CH₃ ofthe 2 diastereomeric Ala overlapping with cyclohexyl aliphatic protons),4.45 (m, 2H, α--H's of cyclohexanol and Ala), 4.95-5.29 (m, 3H, α--H ofAma and Ar--CH₂ of benzyl ester), 5.12 (s, 2H, Ar--CH₂ -- of Z), 6.03(br.d, 1H, urethane H), 6.81, 6.98 (br d, 1H, amide H), 7.32 (s, 10H, 2Ar--H₅).

N-Benzyloxycarbonyl-DL-aminomalonyl(benzyl ester)-D-alanine 1-AdamantylEster (19)

This compound was prepared by the knowndicyclohexylcarbodiimide-hydroxysuccinimide (DCCDOSu) procedure asdescribed for the preparation of compound (18) above on a 20 mmol scale.Acetonitrile was the solvent for this coupling. The crude product (11.2g) was purified on a 600 g silica gel column by gradient elution withtoluene to toluene:EtOAc=9:1. The crude oil so obtained was crystallizedby scratching in hexane in an ice-water bath: yielded 8.66 g (79%). 250MHz nmr (CDCl₃, ppm) 1.19 (d, 1.5H, β--CH₃ of Ala of one of the twodiastereomers, J=7.35 Hz), 1.35 (d, 1.5H, β--CH₃ of Ala of the seconddiastereomer J=6.98 Hz), 1.62 (s, 6H, adamantyl aliphatic protons of C's4, 6 and 10), 2.07 (s, 6H, adamantyl aliphatic protons of C's 2, 8 and9), 2.16 (s, 3H, adamantyl aliphatic protons of C's 3, 5 and 7), 4.23(m, 1H, α--H of Ala), 5.09 (s, 2H, Ar--CH₂ --O-- of Z), 5.17-5.30 (m,3H, Ar--CH₂ --O-- benzyl ester overlapping with α--H of Ama), 7.32 (s,10H, 2 ArH₅).

DL-Aminomalonyl-L-alanine Isopropyl Ester (20) (comparative non-sweetisomers)

This compound was prepared by the procedure as described for theD-alanine isomers. The crude product was recrystallized from MeOH: yield83.5 mg (68%), mp 108°-109°, 250 MHz nmr (CD₃ OD, ppm) 1.25 (m, 6H,(CH₃)₂ CH--O), 1.39 (m, 3H, β--CH₃ of Ala), 4.38 (2q, 1H, α--H of Ala),4.99 (m, 1H, α--H of isopropyl).

Anal. Calcd for C₉ H₁₆ N₂ O₅ : C, 46.55, H, 6.95; N, 12.06; Found C,46.18; H, 7.02; N, 12.11.

DL-Aminomalonyl-D-alanine Fenchyl Ester (21) (sweet)

This compound was prepared by the procedure as described for theisopropyl ester analog. The crude product was recrystallized fromMeOH--H₂ O: yield 0.513 g (67.5%), 250 MHz nmr (CD₃ OD, ppm) 0.80 (m,3H, fenchyl 1--CH₃), 1.06 (m, 3H, fenchyl 2--CH₃), 1.46 (m, 3H, β--CH₃of Ala), 4.37 (m, 1H, α--H of Ala), 4.51 (m, 1H, α--H of fenchylalcohol).

Calcd for C₁₆ H₂₆ N₂ O₅ : C, 58.88; H, 8.03; N, 8.58; Found C, 58.38; H,8.21; N, 8.38.

DL-Aminomalonyl-D-alanine 1-Adamantyl Ester Citrate (22) (sweet)

N-Benzyloxycarbonyl-DL-aminomalonyl-(benzyl ester)-D-alanine 1-adamantylester (0.523 g, 0.955 mmol) and citric acid (0.414 g, 0.955 mmol) weredissolved in 11 mL MeOH and hydrogenated over 54 mg 10% Pd/C at 40 psifor 7-10 minutes. The methanolic solution was then filtered through aCelite pad to give a colorless filtrate, which was concentrated to 2-3mL and precipitated with ether: yield 0.350 g (73%), mp 108°-112°, 250MHz nmr (CD₃ OD, ppm) 1.39 (m, 3H, β--CH₃ of Ala), 1.71 (s, 6H,adamantyl aliphatic protons of C's 4, 6 and 10), 2.13 (s, 9H, adamantylaliphatic protons), 2.72, 2.92 (2d, 4H, --CH₂ --COOH), 4.32 (m, 1H, α--Hof Ala). Nmr indicated 2 moles of the dipeptides combine with 1 mole ofcitric acid.

Calcd for C₃₈ H₅₆ N₄ O₁₇ : C, 54.28; H, 6.71; N, 6.66; Found, C, 54.55;H, 7.00; N, 6.80.

DL-Aminomalonyl-D-alanine trans-2-Methylcyclohexyl Ester Citrate (23)(sweet)

N-Benzyloxycarbonyl-DL-aminomalonyl-(benzyl ester)-D-alaninetrans-2-methylcyclohexyl ester (99.8 mg, 0.195 mmol) was dissolved in 6mL MeOH and hydrogenated over 10.5 mg of 10% Pd/C at atmosphericpressure for 7 minutes until no more H₂ was consumed. The solution wasfiltered through a Celite pad and the colorless filtrate was evaporatedto dryness under reduced pressure to give a white foam. The crudeproduct was quickly dissolved in 6 mL EtOH and citric acid (37.5 mg;0.195 mmol) was added. The solution was evaporated to dryness to give awhite mass. The crude product was recrystallized from EtOH-ether. The 60MHz nmr spectrum of this compound indicated that 2 moles of thedipeptide combine with one mole of citric acid, mp 71°-75°. 250 MHz nmr(CD₃ OD, ppm) of the free dipeptides: 1.11-1.92 (m, 9H, cyclohexanolaliphatic protons), 1.41 (d, 3H, β--CH₃, of Ala partially overlappingwith the aliphatic protons, J=7.35 Hz), 4.12 (m, 2H, α--H of Ala andα--H of trans-2-methylcyclohexanol).

D-Alaninamide Hydrochloride (24)

D-Alanine methyl ester hydrochloride [M. Zaoral et al, Collection Czech,Chem. Commun., 32, 843 (1967)] is converted to the title amide followingthe procedure [R. W. Chambers and F. H. Carpenter, J. Am. Chem. Soc.,77, 1522 (1955)] reported for L-alaninamide hydrochloride [J. K. Changet al, J. Med. Chem., 14, 484 (1971)].

N-Benzyloxycarbonyl-D-alanine Isopropyl Amide (25)

N-Benzyloxycarbonyl-D-alanine (2.232 g, 10 mmol) and HOBt hydrate (1.53g, 10 mmol) are dissolved in 40 mL freshly distilled DMF and cooled inan ice-H₂ O bath. DCC (2.06 g, 10 mmol) is added. The reaction mixtureis stirred in the cold for 15 minutes, isopropylamine (0.85 mL, 10 mmol)is then introduced. It is stirred in the cold for 2 hours and roomtemperature overnight. HOAc (a few drops) is added and the reactionmixture stirred at room temperature for another 15 minutes. It isfiltered to remove DCU and the filtrate layered with 50 mL EtOAc and 80mL H₂ O. The phases are separated and the aqueous phase is extractedwith 50 mL EtOAc. The pooled EtOAc extract is wazhed with H₂ O, 0.1N HCl(3×), H₂ O (3×), 0.1N NaOH (3×), H₂ O (3×), dried over anhydrous Na₂ SO₄and evaporated to dryness. The crude product is purified by liquidcolumn chromatography on silica gel eluting with toluene/EtOAc mixtures.

D-Alanine Isopropyl Amide (26)

N-Benzyloxycarbonyl-D-alanine isopropyl amide (1.322 g, 5 mmol) ishydrogenated in 20 mL MeOH over 100 mg 10% Pd/C in a Parr Apparatus at40 psi and room temperature until no more hydrogen is consumed. Themethanolic suspension is filtered through a Celite pad to remove thecatalyst and the filtrate is evaporated to dryness. The desired productis purified by conversion into a crystalline amino acid amide salt.

N-Benzyloxycarbonyl-D-alanine 1-Adamantyl Amide (27)

The title compound is prepared from N-benzyloxycarbonyl-D-alanine and1-adamantanamine using the usual DCC-HOBt procedure described above.

D-Alanine 1-Adamantyl Amide (28)

N-Benzyloxycarbonyl-D-alanine 1-adamantyl amide is hydrogenated by theprecedure as described for the isopropyl amide.

N-Benzyloxycarbonyl-DL-aminomalonyl(monobenzylester)-D-alanine-Isopropyl Amide (29)

N-Benzyloxycarbonyl-DL-aminomalonic acid monobenzyl ester (0.156 g,0.434 mmol) is dissolved in 1.5 mL DMF. The solution is cooled to -15°to -20° and then treated with DCC (94 mg, 0.455 mmol) and1-hydroxybenzotriazole hydrate (62 mg, 0.46 mmol). The reaction mixtureis stirred at -15° to -20° for 30 minutes. D-Alanine-isopropyl amidehydrochloride (76 mg, 0.46 mmol) is then added followed by the additionof N-methylmorpholine (50 μl, 0.46 mmol). The reaction mixture isstirred at -15° to -20° for 1.5 hours, at 0° for 1 hour and at roomtemperature for 8 hours. The reaction mixture is filtered to remove DCUand the filtrate is freed of solvent under high vacuum. The residue istaken up in EtOAc and filtered. The EtOAc filtrate is then washed with0.1N HCl (3×), H₂ O (3×), 0.1N NaOH (3×), H₂ O (3×), dried overanhydrous Na₂ SO₄ and evaporated to dryness. The crude product is thenchromatographed on silica gel by elution with tolucene/EtOAc mixtures.The product containing fraction as identified by nmr, is precipitatedfrom EtOAc, MeOH and ether mixtures.

N-Benzyloxycarbonyl-DL-aminomalonyl-(monobenzylester)-D-alanine-1-Adamantyl Amide (30)

This compound is prepared by the usual DCC mediate procedure asdescribed for the isopropyl analog. Acetonitrile and THF can also beemployed as the solvent for this coupling. The crude product is purifiedon a silica gel column by elution with toluene/EtOAc mixtures. Theproduct so obtained is crystallized from hexane or precipitation fromEtOAc with ether or hexane.

N-Benzyloxycarbonyl-DL-aminomalonyl(monobenzyl ester)-D-alanamide (31)

N-Benzyloxycarbonyl-DL-aminomalonic acid monobenzyl ester (0.343 g, 1mmol) is dissolved in 4 mL THF and cooled to -15° to -20°. DCC (0.206 g,1 mmol) and N-hydroxysuccinimide (0.115 g, 1 mmol) are then added. Thereaction mixture is stirred in the cold for 30 minutes. D-Alaninamidehydrochloride (0.124 g, 1 mmol) is then added followed by the additionof Et₃ N (0.14 mL, 1 mmol). The reaction mixture is stirred at -15° to-20° for 2.5 hours, 0° for 1 hour and room temperature overnight. A fewdrops of HOAc is added. The reaction mixture is stirred for anadditional 15 minutes. After DCU is removed by filtration, the filtrateis evaporated to dryness. The crude product is purified bychromatography on silica gel by elution with toluene/EtOAc mixtures. Thefraction identified by nmr as the desired product is crystallized orprecipitated from hexane, EtOAc, ether mixtures.

DL-Aminomalonyl-D-alaninamide (32)

N-Benzyloxycarbonyl-DL-aminomalonyl-(benzyl ester)-D-alaninamide (4.13g, 10 mmol) is hydrogenated in 40 mL MeOH over 400 mg 10% Pd/C in a Parrapparatus at room temperature and 40 psi. Products are crystallized fromalcohols-diethyl ether.

DL-Aminomalonyl-D-alanine Isopropyl Amide (33)

N-Benzyloxycarbonyl-DL-aminomalonyl-(benzyl ester)-D-alanine isopropylester is hydrogenated by the procedure described for the D-alaninamideanalog. The product is similarly purified.

DL-Aminomalonyl-D-alanine 1-Adamantyl Amide (34)

This material is prepared by the same hydrogenolysis as described forthe D-alaninamide analog.

II. Taste Test

Many artificial sweeteners do not have a pleasant sucrose-like tastethat is free of detracting side tastes and after tastes. Thesecompounds, including many of the peptides, exhibit side tastes such asbeing minty, or licorice-like, or are even bitter or medicine-like.Others exhibit after tastes such as a metallic taste. Still othersweeteners exhibit a lingering sweet taste. Unlike many artificialsweeteners, the isopropyl ester described in this taste test exhibits anuncommon sucrose-like sweet taste that is free of detracting side andafter tastes. The isopropyl ester exhibits a rapid onset and a rapiddecay of sweet taste, much like sucrose.

Following established methods a taste panel was set up and trained. Thecompound was tasted at concentrations of 0.021, 0.007 and 0.21% w/v(0.86, 3.0 and 9.0 mM) versus aspartame concentrations of 0.01, 0.03,0.1 and 0.3% w/v (0.34, 1.0, 3.4 and 10 mM) and sucrose 2, 4, 9, 18 and36% w/v (62, 125, 250, 500 and 1000 mM). The normalized averaged resultsfor the isopropyl ester, sucrose and aspartame are given in Table 1 andshown graphically in FIG. 2.

Both the isopropyl ester and aspartame solutions were rated as sweet andpleasant by the taste panel. The relative potencies of the sweetenersare expressed as the reciprocal of the concentrations required for ascore of 50 on the sweetness scale. Based on sucrose=1.0, the sweetnesspotency of of the preferred isopropyl ester would be 58 and that ofaspartame would be 126 on a weight scale.

Since the potency response lines are approximately parallel, the abovepotency comparison method is valid.

III. Stability Studies

The most significant limitation of those few peptides here-to-forereported that have good taste qualities is their poor stability inaqueous solution. Those peptides have carboxyl-terminal L-amino acidesters that require for sweetness that the ester is made from a smallalkyl alcohol, most often methanol. Such peptide esters are especiallylabile to hydrolysis and cyclization in aqueous solution resulting inthe generation of non-sweet products. However, the formation of peptideesters of hindered alcohols, such as isopropyl alcohol, for thesecarboxyl-terminal L-amino acid peptides results in peptide esters withmuch reduced or no sweet taste. Thus, for carboxyl-terminal L-amino aciddipeptide esters, sweet taste and poor stability in solution are relatedproperties.

The unexpected discovery of the sweet taste of the carboxyl-terminalD-amino acid dipeptide ester, D,L-aminomalonyl-D-alanine isopropylester, provided an intense peptide ester sweetener exhibiting stabilitythat is markedly superior to the prior art. For example, in pH 3.5solution, the isopropyl ester showed less than 5% degradation after 36days at ambient temperature while after the same period aspartame wasmore than 50% degraded to both more and less polar products. This tenfold improvement in stability is especially significant as regards itsuse in comparably acidic soft drinks. Furthermore, after 36 days in pH7.4 buffer the isopropyl ester was 70% degraded while after onlyone-fourth that time (9 days) aspartame was 100% degraded. These resultsshow that in the key issue of stability of peptide sweeteners, theisopropyl ester offers marked improvements relative to the prior art.

Stability was examined in aqueous solution at various pHs. Solutions (1%w/v) were prepared in 0.05M phosphate buffer adjusted to pH 7.4 and pH3.5. Storage at ambient temperature over 36 days was monitored by hplcanalysis (waters radial-pak reverse phase C-8 column, 25% CH₃ CN, 0.05%TFA/H₂ O eluant, 210 nm U.V. detection). Aspartame was examinedsimilarly for comparison purposes.

IV. Ames/Salmonella Mutagenicity Assay

The isopropyl ester was tested for mutuagenic potential in the Amesassay employing: (1) five Salmonella strains which detect frameshiftmutagens or base-pair substitution mutagens, (2) both the presence andabsence of mammalian S9 microsomal liver fraction as the metabolicactivator, which differentiate promutagens from direct-acting mutagens,(3) five dose levels (3000, 300, 30, 3, 0.3 ug/plate), and (4) threeplates per dose. The compound was also tested for toxicity, with andwithout S9 activation, at the two highest doses. The compound showedneither mutagenic potential nor toxicity for any of these tests.Positive controls and spontaneous background revertants were withinacceptable ranges.

V. Acute Toxicity Testing

The isopropyl ester was evaluated for acute oral toxicity in Swiss malemice. The testing parameters were as follows: each test group containedsix mice (20-30 grams each); the isopropyl ester was administered in anaqueous solution as a single oral dose of 100 mg/kg and 300 mg/kg; twosolvent controls were also administered; the mice were again given foodtwo hours after sample administration.

Body weights were measured on day -3, 0, +7 and +14. Physiologicalparameters were examined the entire first day, then three times a daythrough the first week and twice a day for the second week. Blood serumanalyses for urea nitrogen and alanine aminotransferase activities weremeasured on day -3 and day +14. On day 14, a gross necropsy wasperformed on each mouse.

The results of this acute whole animal toxicity study indicated no toxiceffect under the conditions tested. The toxicological parameters wereall negative and no behavioral or pharmacological effects were observed.There was no excessive eating or drinking during the study. The weightsof all test animals during the 14-day study were normal. No toxiceffects were detected in the necropsy.

Blood samples were collected from the tail vein for biochemical testingof serum samples. This procedure virtually eliminated hemolyzed samples.Neither elevated serum urea nitrogen nor elevated alanineaminotransferase activity were detected in post-study samples fromeither the 100 mg/kg or the 300 mg/kg doses. These analyses demonstratedthe absence of kidney and liver toxicity respectively.

Therefore the isopropyl ester appears to be nontoxic at all doses testedas determined by biochemical acceptance criteria as well.

                                      TABLE 1                                     __________________________________________________________________________    Taste Panel Results                                                           Average Scores for Standard and Test Compounds.sup.1                          Test Series: 4                                                                      Concentration       Off After                                           Compound                                                                            (Millimolar)                                                                          Sweet?                                                                            Bitter?                                                                           Salty?                                                                            Taste?                                                                            Taste?                                                                            Pleasant?                                   __________________________________________________________________________    Aspartame                                                                           0.3392  15  0   1   1   1    4                                                        (7) (0) (1) (1) (1) (2)                                               1.0194  27  0   3   4   2   15                                                        (5) (0) (3) (3) (1) (8)                                               3.3979  68  4   2   2   26  26                                                        (4) (3) (1) (2) (7) (10)                                              10.1937 83  0   1   4   34   9                                                        (6) (0) (1) (4) (14)                                                                              (18)                                        Sucrose                                                                             62.0000 13  3   0   4   0   -4                                                        (7) (3) (0) (4) (0) (8)                                               125.0000                                                                              30  2   8   4   5   18                                                        (5) (1) (5) (3) (2) (9)                                               250.0000                                                                              64  0   l   0   0   49                                                        (3) (0) (1) (0) (0) (8)                                               500.0000                                                                              75  6   12  5   7   32                                                        (4) (5) (9) (3) (3) (11)                                              1000.0000                                                                             97  0   1   0   30  26                                                        (2) (0) (1) (0) (13)                                                                              (20)                                        Isopropyl                                                                           0.8621   4  7   21  13  4   -11                                         Ester         (2) (7) (21)                                                                              (9) (4) (8)                                               3.0172  32  3   5   2   6   13                                                        (8) (3) (5) (2) (4) (8)                                               9.0517  61  1   3   3   4   34                                                        (9) (1) (3) (2) (3) (13)                                        __________________________________________________________________________     .sup.1 The scores have been normalized to 100 and averaged.                   The numbers in parentheses are standard error values.                    

VI. Comparative Taste Test

In order to demonstrate the significant distinction between thecompounds of the present invention having the D-configuration and thatof their isomer having the L-configuration, the following compounds weretested:

Sucrose

Aspartame

Compound (8)-D,L-Aminomalonyl-D-alanine Isopropyl Ester

Compound (21)-D,L-Aminomalonyl-D-alanine Fenchyl Ester

Compound (20)-D,L-Aminomalonyl-L-alanine Isopropyl Ester

The compounds were dissolved in distilled water at the concentrationsspecified in Table 2. Solutions were made up and kept overnight in arefrigerator. The following day, they were poured into individualgraduated plastic cups (10 ml per cup) and allowed to warm to roomtemperature. A taste panel was convened and testing begun that same day.Tasting and evaluation was done by the standard protocol developed byDr. Robert Gesteland of Taste and Smell Consulting Group, Inc.

The first solution tasted (reference standard for the panel) was 0.25Msucrose. Other solutions were tasted in radomized fashion. The randomorder was arrived at as follows:

i. An ordered list of the compounds and concentrations was prepared (26solutions in all).

ii. 0.25M sucrose was designated as 1.

iii. A standard table of random numbers was used in which the numberswere grouped as two-digit numbers. A point in the table was chosen atrandom (blind pointing). The first solution in the list was given thefirst random number in the sequence (read right-to-left, up-to-down)which is between 2 and 26. The second solution in the list was given thenext random number in the sequence between 2 and 26, etc. Repeat numberswere ignored.

iv. Solutions were then labeled A (for number 1) B (for random number2), C-Z.

v. Panelists were instructed to taste the solutions in alphabeticalorder.

With the assistance of Taste and Smell Consulting Group, Inc., tastepanel members were trained adn tested for their ability to discriminatesucrose solutions of varying concentrations. In summary it may be statedthat all 9 panel members had acceptble correlation scores. Their resultswere in good agreement with those which have been previously reported insiilar types of studies. For the test series the correlations for eachpanelist for sucrose and aspartame were acceptable as were the overallresults obtained by averaging individual scores. The results of thetaste panel are set forth in Table 2 wherein the parenthetical figuresrepresent the standard error values for the number above.

Based on the results shown in Table 3, a dose response curve forsucrose, Compound (8) and Compound (20) and same is illustrated in FIG.2.

From a review of FIG. 3, as well as the results set forth in Table 2, itis clear that Compound (8) (the isopropyl ester of the D-configurationaccording to the present invention) is sweet, whereas the correspondingiosmer of the L-configuration, Compound (20), is not sweet.

Compounds (21), (22), and (23) were also found to be sweet.

                                      TABLE 2                                     __________________________________________________________________________    Comparative Taste Panel Results                                               Average Scores for Standard and Test Compounds                                      Concentration       Off After                                           Compound                                                                            (Millimolar)                                                                          Sweet?                                                                            Bitter?                                                                           Salty?                                                                            Taste?                                                                            Taste?                                                                            Pleasant?                                   __________________________________________________________________________    Aspartame                                                                           0.3398  9   1   0   1   1   16                                                        (2) (1) (0) (1) (1) (12)                                              1.0194  27  3   10  6   7    8                                                        (5) (2) (5) (3) (3) (9)                                               3.3979  66  2   3   12  16  22                                                        (4) (2) (3) (6) (5)  (9)                                              10.1937 64  5   0   17  22  12                                                        (10)                                                                              (3) (0) (9) (10)                                                                              (19)                                        Sucrose                                                                             62.0000 3   6   9   9   10  17                                                        (2) (6) (9) (7) (7) (12)                                              125.0000                                                                              30  2   1   3   4   21                                                        (4) (2) (1) (1) (2)  (8)                                              250.0000                                                                              52  3   7   12  15  34                                                        (4) (2) (7) (6) (7)  (9)                                              500.0000                                                                              78  0   0   10  9   35                                                        (4) (0) (0) (6) (5) (11)                                              1000.0000                                                                             95  0   0   12  8   28                                                        (3) (0) (0) (11)                                                                              (5) (19)                                        (8)   0.0431  2   1   1   2   0   15                                                        (2) (1) (1) (2) (0) (12)                                              0.4307  1   12  11  8   7   10                                                        (1) (10)                                                                              (11)                                                                              (6) (6) (13)                                              4.3066  28  0   12  3   5   24                                                        (4) (0) (12)                                                                              (2) (3) (13)                                        (21)  0.0306  1   3   4   13  12  -9                                                        (1) (1) (4) (9) (9)  (6)                                              0.3064  8   29  9   56  50  -57                                                       (5) (9) (6) (12)                                                                              (13)                                                                              (14)                                              3.0637  13  90  62  99  79  -83                                                       (11)                                                                              (8) (19)                                                                              (1) (14)                                                                              (12)                                        (20)  0.0431  3   9   3   5   0   14                                                        (2) (8) (3) (4) (0) (13)                                              0.4307  8   14  16  8   8    5                                                        (7) (10)                                                                              (16)                                                                              (7) (6)  (6)                                              4.3066  5   0   0   5   5   19                                                        (2) (0) (0) (3) (4) (12)                                        __________________________________________________________________________

Although only a few exemplary embodiments of this invention have beendescribed in detail above, those skilled in the art will readilyappreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe following claims.

What is claimed is:
 1. Aminomalonyl-D-alanine derivatives having theformula: ##STR3## and pharmaceutically acceptable salts thereof, whereinX is --NHR or --OR with R being alkyl of from 3 to 10 carbon atoms, or--NH₂.
 2. The derivative of claim 1, wherein R is isopropyl.
 3. Acomposition of matter comprising the combination of at least oneaminomalonyl-D-alanine derivative having the formula: ##STR4## orpharmaceutically acceptable salts thereof, wherein X is --NHR or --ORwith R being alkyl of from 3 to 10 carbon atoms, or --NH₂, dissolved inan aqueous medium.
 4. The composition of claim 3, wherein R isisopropyl.
 5. The composition of claim 3, wherein said aqueous medium isa beverage.
 6. A method of sweetening a beverage, comprising dissolvingtherein at least one aminomalonyl-D-alanine derivative having theformula: ##STR5## or pharmaceutically acceptable salts thereof, whereinX is --NHR or --OR with R being alkyl of from 3 to 10 carbon atoms, or--NH₂, said at least one compound being dissolved in an amountsufficient to effect said sweetening.
 7. The method of claim 6, whereinR is isopropyl.